Natural cyclopeptides from Rubiaceae: advances from the last decade

1 Introduction

Cyclic peptides or cyclopeptides refer to the macromolecules generated by the closing of a peptide chain into a ring structure. Widely distributed in microorganisms, plants, and animals, these compounds play important roles in diverse biological processes and present broad therapeutic potential (Buchanan et al., 2025). Compared to linear peptides, cyclic peptides exhibit enhanced resistance to proteolytic degradation, greater metabolic stability, improved binding affinity and selectivity due to their pre-organized conformation, and, in some cases, favorable cell membrane permeability associated with intramolecular hydrogen bonding, self-assembly capacity, and hydrophobic characteristics (Ding et al., 2026).

Table 1 summarizes the main classes of cyclopeptides according to their biosynthetic origin and structural characteristics. Cyclopeptides are broadly classified into two major groups: ribosomally synthesized and post-translationally modified peptides (RiPPs) and nonribosomal peptides (NRPs are synthesized by dedicated nonribosomal peptide synthetases (NRPSs), large multimodular enzymatic assemblies that produce peptides with remarkable structural and functional diversity) (Süssmuth and Mainz, 2017). RiPPs are genetically encoded as precursor proteins that undergo enzyme-mediated post-translational modifications (Montalbán-López et al., 2021), whereas NRPs are assembled independently of the ribosome by nonribosomal peptide synthetases (NRPSs), enabling the incorporation of both proteinogenic and non-proteinogenic amino acids and generating remarkable structural diversity. Within the RiPP family, cyclotides, PawS-type peptides, and orbitides represent the principal classes of plant cyclic peptides. Although all three are ribosomally synthesized and undergo enzymatic head-to-tail cyclization, they differ markedly in their precursor proteins, cyclization mechanisms, peptide size, and structural features.

TABLE 1. Main structural differences between Rubiaceae species-derived cyclopeptides types.

PawS-type (Preproalbumin with SFTI-like) peptides are a class of gene-encoded, small, disulfide-bonded macrocyclic peptides matured from unusual napin-type albumin precursors (Figure 1) primarily identified in Asteraceae, such as sunflower (Helianthus annuus) seeds (Franke et al., 2016) and Zinnia haageana. These peptides are distinctive because they are co-produced alongside a typical heterodimeric albumin from a single preproalbumin precursor, such as PawS1, which encodes the well-known sunflower trypsin inhibitor 1 (SFTI-1) (Fisher et al., 2020), (Ramalho et al., 2018). Their biosynthesis is driven by a specialized post-translational mechanism known as biosynthetic parallelism, where plant asparaginyl endopeptidases (AEPs) are recruited to perform a transpeptidation reaction. During this process, the AEP targets specific asparagine (Asn) or aspartic acid (Asp) residues to simultaneously cleave the linear precursor and ligate the ends, creating a stable macrocyclic product. While ancestral forms like PawL1 encode only the albumin, PawS-type peptides represent an evolutionary adaptation where the albumin maturation machinery has been repurposed to produce bioactive cyclic peptides (Ramalho et al., 2018).

Rubiaceae is one of the most representative sources of cyclopeptides, particularly cyclotides. The first cyclotide discovered in nature was isolated from Oldenlandia affinis and named kalata B1⁷. Also known as the coffee family, Rubiaceae is the fourth largest family of flowering plants, comprising more than 14,000 species (Razafimandimbison and Rydin, 2024). It is predominantly distributed in tropical regions, with only a few representatives occurring in temperate areas (Ramalho et al., 2018). Rubiaceae is economically important not only as the source of coffee (Coffea spp. L.) but also as a source of biologically active secondary metabolites, such as Cinchona L., the natural source of quinine, which possesses antimalarial properties. In Brazil, the family is represented by approximately 1,417 species distributed across 128 genera, that occur in all Brazilian phytogeographic domains (Razafimandimbison and Rydin, 2024).

More than 400 cyclotides have been sequenced and characterized from several distinct families and species. Recent studies estimate that the Rubiaceae and Violaceae families alone may lead to the identification and characterization of more than 150,000 new cyclopeptides based on genomic annotation (Ramalho et al., 2018). In this context, the present survey aims to clearly and objectively summarize the cyclopeptides reported in the literature for species of the Rubiaceae family over the past decade (2016–2026).

2 Methodology

A bibliographic survey was conducted using SciFinder®, PubMed, Scopus and Web of Science databases. The search employed “cyclotides” as the central term, combined with the descriptors “Rubiaceae,” “cyclic peptides,” “orbitides,” “natural products,” “plants,” and “phytochemistry.” with the aim of prospecting information published over the past decade (2016–2026). The manual curation of these results afforded 215 articles. The results were filtered by exclusion of 38 patents, 89 review articles, and selection of isolation and characterization of cyclopeptides, thus leading to 88 analyzed articles, from which, 36 were focused on isolation and characterization and biosynthesis on Rubiaceae.

3 Results and discussion

Table 2 compiles 33 cyclopeptides reported for the Rubiaceae family between 2016 and 2026, together with the respective species from which they were isolated and characterized. All reported peptides belong to the RiPP class. Even though over 40 cyclopeptides have been approved for medical use (Buchanan et al., 2025), the results revealed that there is still a relatively limited number of publications reporting the isolation and structural characterization of cyclopeptides, stimulating the continuation of research of bioactive cyclopeptides from this family.

TABLE 2. Cyclopeptides from the RiPP class reported from plants belonging to Rubiaceae (2016–2026).

Rubiaceae-type cyclotide.

3.1 Cyclotides

Cyclotides are characterized by a cyclic cystine knot containing six conserved cysteine residues and three disulfide bonds, PawS-type peptides originate from unusual napin-type albumin precursors and are typically stabilized by a single disulfide bond, whereas orbitides are small homodetic cyclic peptides that lack disulfide bridges and are generally enriched in hydrophobic amino acids. These biosynthetic and structural differences largely determine their stability, molecular diversity, and biological functions (Zhang et al., 2021; Rajendran et al., 2023).

Our current understanding of plant peptide macrocyclization is largely restricted to head-to-tail cyclic peptides. These metabolites are biosynthesized as RiPPs and undergo a final protease-catalyzed macrocyclization step to generate their mature cyclic structures. By comparison, the genetic determinants and biochemical pathways governing the biosynthesis of side-chain-macrocyclic peptides remain poorly understood and represent a significant knowledge gap in plant peptide biology (Chigumba et al., 2022).

Cyclotides consist of peptide chains containing 28 to 37 amino acid residues arranged in a head-to-tail cyclic structure, formed by a linkage between the N-terminal amino group and the C-terminal carboxyl group. In addition, they contain six conserved cysteine residues organized into three disulfide bonds, which also confer high conformational stability (Zhang et al., 2021). Furthermore, cyclotides can be subdivided into two major subfamilies: Möbius or Bracelet, depending on the presence or absence of a specific amino acid residue in loop 5. This structural difference results in an approximately 180° twist in the peptide backbone conformation, directly impacting the characteristic three-dimensional structure of this class of polypeptides (Rajendran et al., 2023). Beyond this topological distinction, these subfamilies also differ in loop length and sequence composition, as well as in their overall net charge. Bracelet cyclotides generally exhibit a higher positive charge due to the occurrence of Lys and/or Arg residues in loops five and 6, whereas most Möbius cyclotides are neutral and contain relatively few charged amino acid residues. A third subfamily, the trypsin inhibitor cyclotides, was initially discovered in species of the Cucurbitaceae family. Although these peptides share limited sequence homology with the Möbius and bracelet cyclotides, they possess the characteristic cyclic cystine knot (CCK) motif and are biosynthesized through a similar pathway, supporting their inclusion within the cyclotide family. In addition, trypsin inhibitor cyclotides are generally more hydrophilic than members of the Möbius and bracelet subfamilies (Ravipati et al., 2017). A representative examples of cyclotide could be found in Figure 2.

Seven cyclotides (1–7) were isolated from Carapichea ipecacuanha (Falanga et al., 2022). One of them, caripe 8 (2), showed strong antagonistic activity toward the receptor responsible for corticotropin hormone release. Carapichea ipecacuanha, popularly known as “ipeca,” is an herbaceous species with emetic properties, as well as expectorant and amoebicidal activities associated with the presence of alkaloids such as emetine and cephaeline (Falanga et al., 2022; Ferreira et al., 2023).

From Galianthe thalictroides, a species native to central-western Brazil, a novel cyclopeptide named 5-β-hydroxy-RA-III (8) was isolated (Figueiredo et al., 2016). Cyclotide 8 was formally classified as a “Rubiaceae-type cyclotide” (RAs) due to the special configuration of a homobicyclichexapeptidic structure composed of two alanine residues (D-α-alanine and L-α-alanine) containing three N-methyltyrosine residues (Hu et al., 2019; Murthy et al., 2023). Galianthe thalictroides has been traditionally used for the treatment and prevention of cancer. The literature reports the isolation of steroids, iridoid glycosides, alkaloids, and flavonoids from this species (Fahradpour et al., 2017).

Rajendran and co-workers (Rajendran et al., 2023) conducted a screening of more than 50 traditional medicinal plant species in search of new cyclopeptides. Their efforts led to the isolation of one new cyclotide (10) present in Geophila repens with antibiotic and cytotoxic potential. Geophila repens is commonly found in tropical regions of Asia, Africa, the Americas, and Micronesia. Its fruits have also been reported to possess antifungal properties. Several classes of compounds have been described for this genus, particularly terpenes such as β-caryophyllene, myrcene, and friedelin (Rajendran et al., 2023).

Oldenlandia is a large genus of the family Rubiaceae and is widely recognized for its medicinal value, being traditionally used in the treatment of skin diseases, dysentery, wounds, snakebites, and cancer, among other conditions (Saranya et al., 2024). Species belonging to this genus are known to produce a wide range of bioactive secondary metabolites, including iridoids, anthraquinones, triterpenes, and coumarins, among others (Divya et al., 2023; Al-Shuhaib and Al-Shuhaib, 2024). The fungus Aspergillus niger was able to enhance cyclopeptide production in Oldenlandia umbellate. After peptide sequencing, one cyclotides cycloviolacin O23 (14) was identified (Saranya et al., 2024).

Pinto and co-workers also isolated and characterized both the cyclotide parigidin-br-1–3 (18) and its acyclotide counterpart (parigidin-br 3) from P. rigida (Pinto et al., 2016). P. rigida can be found in Central and South America, and it is commonly known as “gritadeira”. It is a medicinal plant used to treat mostly urinary disorders (Dos Santos et al., 2021). Acyclotides, similarly to cyclotides, contain cystine knots and exhibit a high degree of amino acid sequence homology with conventional cyclotides but are biosynthetically unable to undergo cyclization (Pinto et al., 2016). Few natural acyclotides have been isolated from plants, however, studies indicate that most arise from deleterious mutations involving asparagine and aspartic acid residues, which play a crucial role in the biosynthetic process (Pinto et al., 2016). In other work by the same group with the leaves and stems of Palicourea sessilis the cyclotide kBS (19) was also isolated.

One of the most important genera in the study of natural cyclotides within the Rubiaceae family is Rubia, which comprises more than 60 species widely distributed across Africa, Asia, the Americas, and Europe (Murthy et al., 2023). Cyclotides found in this genus were usually referred to as RAs. Around 30 RAs were isolated from nine Rubia species (R. cordifolia, R. yunnanensis, R. schumanniana, R. alata, R. wallichiana, R. oncotricha, R. sylvatica, R. ovatifolia, and Rubia podantha), including rubipodanin B, an unreported compound (Hu et al., 2019; Murthy et al., 2023). Seven of the twelve compounds (26–33) exhibited cytotoxicity against three human cancer cell lines (MDA-MB-231, SW-620, and HepG2), and two showed strong binding affinity toward the NF-κB transcription protein complex. The 2-alanine amino acid residues are essential for the observed biological activities (Hu et al., 2019).

3.2 Orbitides

Orbitides are a class of small, gene-encoded plant cyclic peptides, typically ranging from 5 to 16 amino acid residues in length. Figure 2 presents three representative orbitides isolated from members of the Rutaceae, Asteraceae, and Euphorbiaceae families, as no experimentally determined three-dimensional structures of orbitides are currently available in the Protein Data Bank (PDB). Formerly known as Caryophyllaceae-type or Type VI cyclopeptides, they are defined as ribosomally synthesized and post-translationally modified peptides (RiPPs) that are head-to-tail cyclized via an N-to-C terminal amide bond. Unlike cyclotides, orbitides are homodetic cycles that lack disulfide bonds and are frequently characterized by a high proportion of hydrophobic residues (Razafimandimbison and Rydin, 2024; Daly and Wilson, 2021; Pinto et al., 2021).

Studies employing synthetic peptide substrates and seed extracts from Saponaria vaccaria demonstrated that orbitides are initially synthesized as linear precursor peptides. Presegetalin A1, a 32-amino acid precursor, undergoes a sequential enzymatic maturation process involving two key enzymes. First, the serine protease OLP1 cleaves the N-terminal leader region, releasing an intermediate bearing an N-terminal glycine residue. Subsequently, the peptide cyclase PCY1 removes the C-terminal recognition sequence and concomitantly catalyzes head-to-tail macrocyclization, yielding the mature orbitide segetalin A, c (Gly-Val-Pro-Val-Trp-Ala). This two-step proteolytic mechanism represents one of the best-characterized biosynthetic pathways for plant orbitides (Ramalho et al., 2018).

They have been identified in a diverse range of plant families, including Rutaceae, Euphorbiaceae, Caryophyllaceae, and Linaceae, and exhibit various biological activities such as antibacterial, antifungal, and immunosuppressive properties. The maturation of orbitides involves a complex biosynthetic process where precursor proteins are cleaved and cyclized by specialized enzymes, such as prolyl oligopeptidases or other yet-to-be-identified proteases (Razafimandimbison and Rydin, 2024; Daly and Wilson, 2021; Pinto et al., 2021).

Like cyclotides and other RiPPs, orbitides are biosynthesized through convergent pathways that employ specialized serine proteases capable of catalyzing transamidation, thereby favoring macrocyclization over peptide hydrolysis. Studies on Citrus species (Rutaceae) further corroborated this biosynthetic model, demonstrating that orbitides arise via a ribosome-dependent mechanism involving cyclopeptide precursor proteins (CPPs). The identification of genes encoding these precursor peptides provided compelling molecular evidence for the genetic basis of orbitide biosynthesis and reinforced their classification as RiPP natural products (Ramalho et al., 2018).

The orbitide 6-O-methylbouvardin (9) was initially isolated from Bouvardia ternifolia and G. thalictroides (Falanga et al., 2022). From G. repens, the orbitides (11–13) were also isolated by Rajendran and collaborators further expanding the chemical diversity of cyclopeptides for the Rubiaceae family (Rajendran et al., 2023).

Another noteworthy source of orbitides is Oldenlandia umbellata, from which Saranya et a¹⁹. Isolated the orbitide Varv-A (15). In addition, Rupachandra and Bertilla (Bertilla and Rupachandra, 2026) identified two Kalata-type cyclotides, designated Kalata B8-type and Kalata B2-type, by LC-MS/MS analysis of a cyclotide-rich fraction obtained from the same species.

Plants of the genus Ophiorrhiza are widely distributed throughout tropical and subtropical regions and have long been used in traditional medicine for the treatment of snakebites, ulcers, and skin disorders. From Ophiorrhiza nutans, Onozawa et al. (Onozawa et al., 2017) isolated the orbitide ophiorrhisine A (16), a unique tetrapeptide characterized by the presence of a 14-membered paracyclophane ring and an N,N,N-trimethyltyrosine residue in its side chain. However, this compound did not exhibit significant cytotoxic activity.

The genus Palicourea is particularly recognized for its ability to biosynthesize monoterpene indole alkaloids and glycosylated monoterpene indole alkaloids (Pinto et al., 2016; Samulski et al., 2020). Pinto et al. (Ramalho et al., 2018) isolated the orbitide palicourein (17) from Palicourea condensata and subsequently investigated the leaves and stems of P. sessilis, leading to the characterization of five new orbitides, pase A–E (20–24). In addition to cyclopeptides, the aerial parts of P. sessilis have been reported to contain terpenoids such as squalene, stigmasterol, and lupeol, as well as quercetin and its derivatives (Fisher et al., 2020). The same authors also reported the isolation of the orbitide paltet-1 (25) from Palicourea tetragona (Ramalho et al., 2018).

Collectively, these findings further emphasize the importance of the Rubiaceae family as a rich and still underexplored source of structurally diverse and biologically active cyclopeptides, particularly orbitides with potential pharmacological applications.

4 Conclusion

In this mini-review, we highlight the most relevant findings reported over the last 10 years regarding natural cyclopeptides from Rubiaceae. Although this review does not encompass all publications addressing the topic, it clearly demonstrates the extensive biochemical potential of this class of compounds and the potential of this botanical family. Cyclopeptides are special mainly due to their typically rigid structures, favorable bioavailability and cell permeability, stability, and specificity. Despite this, the number of studies published over the past decade indicates that this field of research remains relatively underexplored. Despite the increasing interest in plant cyclopeptides, their isolation and structural elucidation remain major challenges in natural products chemistry. The generally low concentrations of these metabolites in plant tissues, coupled with the chemical complexity of the extracts, hinder their detection, isolation, and purification. In addition, the cyclic architecture of these peptides complicates the unequivocal assignment of the cyclization site and the differentiation of structurally related analogues, which often vary only by a single amino acid substitution or residue arrangement. Because these congeners usually possess very similar physicochemical properties, their chromatographic resolution is frequently difficult, making the purification of individual compounds particularly challenging. However, with the implementation of emerging technologies such as transcriptomics it is expected that new cyclopeptides (particularly with respect to PawS-derived cyclopeptides, which, to date, have not been reported in species belonging to the Rubiaceae family) with potential applications for human benefit will be discovered with increasing speed and accuracy.

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Footnotes

Edited by: Lucia Pinheiro Santos Pimenta, Federal University of Minas Gerais, Brazil

Reviewed by: Suze Ma, Zhejiang University, China